Jong Cheol Lee1, Won Hyeok Lee2, Young Joo Min3, Hee Jeong Cha4, Myung Woul Han5, Hyo Won Chang6, Sun-A Kim7, Seung-Ho Choi6, Seong Who Kim8, Sang Yoon Kim9. 1. Department of Otorhinolaryngology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea; Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea. 2. Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea. 3. Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea; Department of Internal Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea. 4. Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea. 5. Department of Otorhinolaryngology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea. 6. Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. 7. Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. 8. Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. Electronic address: swhokim@gmail.com. 9. Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea. Electronic address: sykim3715@gmail.com.
Abstract
PURPOSE: There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis. METHODS AND MATERIALS: Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line. RESULTS: HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine. CONCLUSION: Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.
PURPOSE: There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis. METHODS AND MATERIALS: Using a previously established HN3 cell line from a laryngeal carcinomapatient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line. RESULTS: HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine. CONCLUSION: Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.
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Authors: Won Hyeok Lee; Myung Woul Han; Song Hee Kim; Daseul Seong; Jae Hee An; Hyo Won Chang; Sang Yoon Kim; Seong Who Kim; Jong Cheol Lee Journal: Cells Date: 2020-08-07 Impact factor: 6.600